Cellular Fractionation and Protein Detection

For separation of total cellular fraction, cells were lysed with lysis buffer (50 mM NaCl, 1% Nonidet P-40 and 50 mM Tris-HCl, pH 8.0) containing protease inhibitor cocktail (Nacalai Tesque, Inc.). For separation of cytoplasmic and nuclear fractions, cells were lysed in the fractionation buffer [phosphate-buffered saline containing 0.1% Nonidet P-40 and the protease inhibitor cocktail (Nacalai Tesque, Inc.)]and centrifuged at 15,000 × g for 10 sec at 4°C to obtain the cytosolic fraction (supernatants). The insoluble pellets were resuspended in the fractionation buffer and centrifuged at 15,000 × g for 10 sec at 4°C to obtain the nuclear fraction (pellets). Either glyceraldehyde 3-phosphate dehydrogenase (Gapdh) or fibrillarin (Fbl) was used as the cytoplasmic or nuclear marker protein, respectively (26 (link)).